The project will apply induced pluripotential stem cell (iPS) technology to understand the process of limb regeneration in a model organism. Tadpoles of the frog Xenopus laevis are normally able to regenerate their limbs following amputation at early developmental stages but not at later stages. This situation provides a potential gain-of-function assay for factors promoting limb regeneration. My central hypothesis is that regeneration of Xenopus froglet or late stage tadpole limbs will occur if the limb fibroblasts can be respecified to an early tadpole limb bud phenotype. This will be achieved by introducing specific transcription factors into froglet limb cells cultured in vitro. Fibroblasts will be cultured from limbs of froglets that carry an antibiotic resistance gene driven by a limb bud- specific promoter: prx1. Transcription factors that encode limb bud status will then be introduced using inducible lentivirus, in the presence of treatments or other genes that will open chromatin and enable the transcription factors to locate their target genes in the DNA. Cells that are respecified to the early limb bud state will activate the prx1 promoter and will then be isolated by antibiotic selection. The cells will be characterized by expression analysis for other limb bud genes. A new assay for regeneration-competence will be refined. This is done by injecting limb bud cells subcutaneously into froglet hindlimbs, allowing to heal, amputating through the graft region, and then assessing the degree of regeneration achieved. This method will be refined using regeneration-competent tadpole limb cells for the graft, and will then used to test the regenerative capacity of the cells modified in culture. The use of intrinsic genetic markers in the donor cells will enable us to detect whether the regenerate has arisen from donor cells, host cells, or a mixture of the two, and whether there are positional information type interactions between graft and host affecting the pattern of the regenerated limbs. This project is innovative in several ways. Firstly it is the first time that iPS technology has been applied to a problem of regeneration. Secondly, it applies tissue culture to a regeneration problem, which has seldom been done before. Thirdly it makes use of selective techniques to obtain the desired cells, again a technique that has not previously been used in regeneration research. The progressive loss of Xenopus tadpole limbs to regenerate is correlated with an inability to re-activate expression of early limb bud genes. This is also characteristic of mammalian limbs. Once developed, the proposed method is therefore expected also to be capable of stimulating regeneration in mammalian limbs. PUBLIC HEALTH RELEVANCE: Early tadpole legs will regenerate after they are cut off, later ones will not. This innovative proposal will use modern stem cell technology to test a hypothesis about why this is so. If the hypothesis is confirmed, the methods developed should assist in facilitating the regeneration of mammalian limbs.